Selective Blocker Research Articles

Impaired Endothelial Vasomotor Function with Borderline-High Triglycerides

Approximately 1 in 3 adults in the United States have a triglyceride (TG) concentration above 150 mg/dL and 1 in 5 adults have TG levels 200 mg/dL or higher. Moreover, the prevalence of suboptimal TGs (>150 mg/dL) has increased 5-fold in middle-aged and older adults over the last 3 decades. While much attention has focused on the cardiovascular effects of high and very high TG levels, where other lipid and metabolic abnormalities are often present, there are currently little data on the influence of borderline-high TG concentrations on vascular health. This represents a critical void in our understanding of the pathogenesis of cardiovascular disease (CVD) with TGs. Impaired endothelial vasomotor function is a central etiologic feature in the development of CVD and associated thrombotic events. We tested the hypothesis that endothelium-dependent vasomotor function is impaired in adults with borderline-high TG concentrations. Thirty-two sedentary, adults were studied: 15 (7M/8F, age: 56±1 yr; BMI: 26.8±0.6 kg/m2) with normal TG (35-140 mg/dL) and 17 (9M/8F, age: 57±1 yr; BMI: 28.7±0.8 kg/m2) with borderline-high TG (150-199 mg/dL) levels. Forearm blood flow (FBF; plethysmography) was determined in response to the intra-arterial infusion: acetylcholine (ACh) (4.0, 8.0, and 16.0 mg/100 mL tissue/min), sodium nitroprusside (1.0, 2.0, and 4.0 mg/100 mL tissue/min) and selective ETa receptor blocker (BQ-123, 100 nmol/min for 60 min). FBF response to ACh was significantly lower (~20%) in the borderline-high TG (from 4.7±0.3 to 11.6+0.6 mL/100 mL tissue/min) vs normal TG (from 4.3±0.3 to 14.6±0.5 mL/100 mL tissue/min) group. There were no significant group differences in the FBF responses to sodium nitroprusside. BQ-123 elicited a significant vasodilator response (~20%) in the borderline-high TG adults but did not alter FBF in the normal TG adults. Co-infusion of BQ-123 did not affect FBF responses to ACh in normal TG group but resulted in an ~30% increase in Ach-induced vasodilation (from 4.3±0.4 to 15.1±0.7 mL/100 mL tissue/min; P<0.05) in borderline-high TG group. Triglyceride levels were significantly and inversely associated with total FBF response to ACh (r=-0.63) and positively associated with the peak FBF response to BQ-123 (r=0.53; P<0.05). These results indicate that borderline-high TG concentrations are associated with diminished endothelium-dependent vasodilation and enhanced ET-1-mediated vasoconstrictor tone. Impaired endothelial vasomotor function may underlie the increased risk of CVD in adults with borderline-high TG levels. Nothing to disclose This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Study on the Analgesic Activity of Peptide from Conus achates.

As a peptide originally discovered from Conus achates by mass spectrometry and cDNA sequencing, Ac6.4 contains 25 amino acid residues and three disulfide bridges. Our previous study found that this peptide possesses 80% similarity to MVIIA by BLAST and that MVIIA is a potent and selective blocker of N-type voltage-sensitive calcium channels in neurons. To recognize the target protein and analgesic activity of Ac6.4 from Conus achates. In the present study, we synthesized Ac6.4, expressed the Trx-Ac6.4 fusion protein, tested Ac6.4 for its inhibitory activity against Cav2.2 in CHO cells and investigated Ac6.4 and Trx-Ac6.4 for their analgesic activities in mice. Data revealed that Ac6.4 had strong inhibitory activity against Cav2.2 (IC50 = 43.6 nM). After intracranial administration of Ac6.4 (5, 10, 20 μg/kg) and Trx-Ac6.4 (20, 40, 80 μg/kg), significant analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent. This study expands our knowledge of the peptide Ac6.4 and provides new possibilities for developing Cav2.2 inhibitors and analgesic drugs.

Bidirectional TRP/L Type Ca2+ Channel/RyR/BKCa Molecular and Functional Signaloplex in Vascular Smooth Muscles.

TRP channels are expressed both in vascular myocytes and endothelial cells, but knowledge of their operational mechanisms in vascular tissue is particularly limited. Here, we show for the first time the biphasic contractile reaction with relaxation followed by a contraction in response to TRPV4 agonist, GSK1016790A, in a rat pulmonary artery preconstricted with phenylephrine. Similar responses were observed both with and without endothelium, and these were abolished by the TRPV4 selective blocker, HC067047, confirming the specific role of TRPV4 in vascular myocytes. Using selective blockers of BKCa and L-type voltage-gated Ca2+ channels (CaL), we found that the relaxation phase was inducted by BKCa activation generating STOCs, while subsequent slowly developing TRPV4-mediated depolarisation activated CaL, producing the second contraction phase. These results are compared to TRPM8 activation using menthol in rat tail artery. Activation of both types of TRP channels produces highly similar changes in membrane potential, namely slow depolarisation with concurrent brief hyperpolarisations due to STOCs. We thus propose a general concept of bidirectional TRP-CaL-RyR-BKCa molecular and functional signaloplex in vascular smooth muscles. Accordingly, both TRPV4 and TRPM8 channels enhance local Ca2+ signals producing STOCs via TRP-RyR-BKCa coupling while simultaneously globally engaging BKCa and CaL channels by altering membrane potential.

The effect of Ranolazine on random pattern skin flap survival in rats

Objective: Flap surgery is widely used in the field of plastic surgery for defect reconstruction. The most important problem of this surgery is ischemic necrosis which may occur in the distal of the flap. For this reason, studies have been conducted to develop many treatment methods to solve this problem. Our study aimed to investigate the effects of different doses of ranolazine, a selective late sodium current blocker, on the viability of skin flaps prepared from the back of the rat. Material and Methods: A total of 32 rats, including 8 rats in each group, were included in the study. A random patterned caudal base 3x9 cm width : length modified McFarlane skin flap was elevated from the backs of all rats and adapted back to its place For 7 days postoperatively, 1 cc of saline was added to the control group (Group A), and 45 mg/kg, 90 mg/kg, and 180 mg/kg of ranolazine were added to the study groups (Group B, C, D), respectively, and administered daily with gastric lavage On the 7th postoperative day, photographs were taken to evaluate the viability of the flaps and scintigraphic evaluations were made. After the rats were sacrificed, the flaps were separated from their bases and evaluated histopathologically. Results: As a result of the evaluations, the viable flap area was calculated as 12.7548 cm2 for Group A, 14.9533 cm2, 16.494 cm2 and 16.7599 cm2 for Groups B, C and D, respectively, and a statistically significant difference was found between the groups (P = 0.00 <0.05 ). The viable flap area percentages were calculated as 57.486% for Group A, 68.908% for Group B, 70.174% for Group C and 74.603% for Group D, and a statistically significant difference was found between the groups (P = 0.00 <0.05). As a result of scintigraphic evaluation, the viable flap area was found to be 61.57% for Group A, 71.04% for Group B, 70.21% for Group C and 73.85% for Group D, and a statistically significant difference was found between the groups (P = 0.03 <0.05). Histopathological evaluation revealed a decrease in the flaps' inflammation, edema and necrosis scores with the increasing drug dose. Conclusion: As a result, although flap viability increased with increasing doses in the group given ranolazine, more detailed studies are needed before it can be used clinically.

Behavioral profile of adult zebrafish acutely exposed to a selective dopamine uptake inhibitor, GBR 12909.

The dopamine transporter (DAT) is the main regulator of dopamine concentration in the extrasynaptic space. The pharmacological inhibition of the DAT results in a wide spectrum of behavioral manifestations, which have been identified so far in a limited number of species, mostly in rodents. Here, we used another well-recognized model organism, the zebrafish (Danio rerio), to explore the behavioral effects of GBR 12909, a highly-affine selective DAT blocker. We evaluated zebrafish locomotion, novelty-related exploration, spatial cognition, and social phenotypes in the novel tank, habituation and shoaling tests, following acute 20-min water immersion in GBR 12909. Our findings show hypolocomotion, anxiety-like state, and impaired spatial cognition in fish acutely treated with GBR 12909. This behavioral profile generally parallels that of the DAT knockout rodents and zebrafish, and it overlaps with behavioral effects of other DAT-inhibiting drugs of abuse, such as cocaine and D-amphetamine. Collectively, our data support the utility of zebrafish in translational studies on DAT targeting neuropharmacology and strongly implicate DAT aberration as an important mechanisms involved in neurological and psychiatric diseases.

Connexin 43 Promotes Neurogenesis via Regulating Aquaporin-4 after Cerebral Ischemia.

We aimed to test the effects of connexin43 (Cx43) on ischemic neurogenesis and examined whether it was dependent on aquaporin-4 (AQP4). We detected the expression of Cx43 and AQP4 in the ipsilateral subventricular zone (SVZ) and peri-infarct cortex after middle cerebral artery occlusion (MCAO). Also, we examined neurogenesis in the above regions via co-labeling of 5-bromo-2-deoxyuridine (BrdU)/neuronal nuclear antigen (NeuN) and BrdU/doublecortin (DCX). The effects of Cx43 and AQP4 were investigated by using two transgenic animals: heterozygous Cx43 (Cx43±) mice and AQP4 knockout (AQP4-/-) mice, and connexin mimetic peptide (CMP), a selective Cx43 blocker. We demonstrated AQP4 and Cx43 were co-expressed in the astrocytes after MCAO and the expression was highly increased in ipsilateral SVZ and peri-infarct cortex. Cx43± mice had larger infarction volumes and worse neurological function. Both BrdU/NeuN and BrdU/DCX co-labeled cells in the two regions were reduced in Cx43± and AQP4-/- mice compared to wild-type (WT) mice, suggesting Cx43 and AQP4 participated in neurogenesis of neural stem cells. Moreover, CMP decreased AQP4 expression and inhibited neurogenesis in WT mice, while the latter failed to be observed in AQP4-/- mice. Besides, higher levels of IL-1β and TNF-α were detected in the SVZ and peri-infarct cortex of AQP4-/- and Cx43± mice than those in WT mice. In conclusion, our data suggest that Cx43 elicits neuroprotective effects after cerebral ischemia through promoting neurogenesis in the SVZ to regenerate the injured neurons, which is AQP4 dependent and associated with down-regulation of inflammatory cytokines IL-1β and TNF-α.

Adeno-Associated virus 8 delivers an immunomodulatory peptide to mouse liver more efficiently than to rat liver.

Targeting the Kv1.3 potassium channel has proven effective in reducing obesity and the severity of animal models of autoimmune disease. Stichodactyla toxin (ShK), isolated from the sea anemone Stichodactyla helianthus, is a potent blocker of Kv1.3. Several of its analogs are some of the most potent and selective blockers of this channel. However, like most biologics, ShK and its analogs require injections for their delivery, and repeated injections reduce patient compliance during the treatment of chronic diseases. We hypothesized that inducing the expression of an ShK analog by hepatocytes would remove the requirement for frequent injections and lead to a sustained level of Kv1.3 blocker in the circulation. To this goal, we tested the ability of Adeno-Associated Virus (AAV)8 vectors to target hepatocytes for expressing the ShK analog, ShK-235 (AAV-ShK-235) in rodents. We designed AAV8 vectors expressing the target transgene, ShK-235, or Enhanced Green fluorescent protein (EGFP). Transduction of mouse livers led to the production of sufficient levels of functional ShK-235 in the serum from AAV-ShK-235 single-injected mice to block Kv1.3 channels. However, AAV-ShK-235 therapy was not effective in reducing high-fat diet-induced obesity in mice. In addition, injection of even high doses of AAV8-ShK-235 to rats resulted in a very low liver transduction efficiency and failed to reduce inflammation in a well-established rat model of delayed-type hypersensitivity. In conclusion, the AAV8-based delivery of ShK-235 was highly effective in inducing the secretion of functional Kv1.3-blocking peptide in mouse, but not rat, hepatocytes yet did not reduce obesity in mice fed a high-fat diet.

A Selective Mineralocorticoid Receptor Blocker, Esaxerenone, Attenuates Vascular Dysfunction in Diabetic C57BL/6 Mice.

Pharmacological blockade of mineralocorticoid receptors (MRs) is a potential therapeutic approach to reduce cardiovascular complications since MRs play a crucial role in cardiovascular regulation. Recent studies suggest that MR antagonists affect several extrarenal tissues, including vessel function. We investigated the effect of a novel nonsteroidal selective MR blocker, esaxerenone, on diabetes-induced vascular dysfunction. Diabetes was induced by a single dose of streptozotocin in 8-week-old male C57BL/6 mice. Esaxerenone (3 mg/kg/day) or a vehicle was administered by gavage to diabetic mice for 3 weeks. Metabolic parameters, plasma aldosterone levels, and parameters related to renal function were measured. Endothelium-dependent or -independent vascular responses of the aortic segments were analyzed with acetylcholine or sodium nitroprusside, respectively. Human umbilical vein endothelial cells (HUVECs) were used for the in vitro study. Induction of diabetes elevated plasma aldosterone level (P＜0.05) and impaired endothelium-dependent vascular relaxation (P＜0.05). The administration of esaxerenone ameliorated the endothelial dysfunction (P＜0.01) without the alteration of metabolic parameters, blood pressure, and renal function. Esaxerenone improved the eNOSSer1177 phosphorylation in the aorta obtained from diabetic mice (P＜0.05) compared with that in the vehicle-treated group. Furthermore, a major MR agonist, aldosterone, decreased eNOSSer1177 phosphorylation and increased eNOSThr495 phosphorylation in HUVECs, which recovered with esaxerenone. Esaxerenone ameliorated the endothelium-dependent vascular relaxation caused by aldosterone in the aortic segments obtained from C57BL/6 mice (P＜0.001). Esaxerenone attenuates the development of diabetes-induced endothelial dysfunction in mice. These results suggest that esaxerenone has potential vascular protective effects in individuals with diabetes.

Initial management of lower urinary tract symptoms and bladder outlet obstruction

Lower urinary tract symptoms (LUTS) are a common presenting complaint for both men and women, affecting >60% of men and women aged >40 years. They encompass a wide range of aetiologies and can be an indicator of covert underlying pathologies such as obesity and metabolic syndrome. The initial assessment should focus on identifying the underlying cause and assessing the impact on quality of life aided by validated objective questionnaires. Risk factors have been identified that can predict the likelihood of progression of LUTS (e.g. urinary retention), such as age, moderately elevated prostate-specific antigen and prostate size. Initial management includes conservative and medical management, and not all patients require surgery. Identifying those at high risk of acute urinary retention helps in counselling and subsequent management decisions. Aside from lifestyle modification, medications include selective α-adrenoceptor blockers, 5α-reductase inhibitors, antimuscarinics, β3-agonists and phosphodiesterase-5 inhibitors, either as monotherapy or combination therapy.

333 Intraventricular Ziconotide Improves Cognitive Outcomes following Lateral Fluid Percussion Injury in a Rat Model

INTRODUCTION: Traumatic brain injury (TBI) is a major source of morbidity and mortality worldwide. In previous studies, intravenous ziconotide, a selective N-type voltage-gated calcium channel blocker, reduced brain calcium accumulation, improved brain mitochondrial respiration, and improved cognitive and motor outcomes in rodent models of TBI. Attempts to translate intravenous ziconotide therapy to humans were halted due to peripheral side-effects. However, ziconotide is now FDA-approved for treatment of severe pain syndromes when administered intrathecally instead of intravenously. We hypothesize that following TBI intraventricular ziconotide will improve cognitive function in the absence of significant side effects. METHODS: Seventy-seven Sprague-Dawley rats underwent sham (n = 11 males, n = 11 females) or lateral fluid percussion (LFP) injury (n = 21 males, n = 23 females). One-hour post-injury, rats received either an intraventricular injection of saline (n = 22 sham, n = 23 LFP) or ziconotide (n = 21 LFP). A catheter was implanted in the left lateral ventricle and connected to an Alzet micro-osmotic pump to allow for 72 hours of continuous infusion. Cognitive outcomes were evaluated using Morris water maze (MWM; days 10-15). RESULTS: TBI-saline rats performed significantly worse than shams on MWM (p < 0.01). Ziconotide treated rats had an intermediate phenotype on MWM. TBI-saline rats swam a less efficient path than sham animals (p < 0.01); ziconotide path efficiency was not significantly different from shams. Performance on the MWM was likely not related to motor deficits as there was no significant effect of group on mean swimming speed in either males or females. Female sham animals performed significantly better on probe trials compared to TBI-saline (p < 0.05); ziconotide animals were not different from shams. CONCLUSIONS: These findings support centrally delivered Ziconotide as a potential neuroprotective agent to improve cognitive performance following TBI. Further studies are required to characterize the neuroprotective effects of Ziconotide therapy.

DEVELOPMENT AND VALIDATION OF HPLC-METHOD FOR SIMULTANEOUS QUANTITATIVE DETERMINATION OF IMIDACLOPRID AND IVERMECTIN IN DROPS AGAINST FLEAS AND TICKS

Imidacloprid and ivermectin are selective blockers of metabotropic ion receptors of the nervous system of invertebrates, leading to paralysis and death of ecto- and endoparasites. These active substances are used in insect-acaricidal drops to kill lice, fleas and ticks in cattle, domestic animals and pets.
 The aim of the work was to develop a method for the identification and simultaneous quantitative determination of imidacloprid and ivermectin in drops for external use. The method was developed and validated by selectivity, linearity and suitability parameters of the chromatographic system. Drops for external use were used as a sample-object for development. The standard sample and the test sample were dissolved in acetonitrile to a concentration of imidacloprid 100 μg/ml and ivermectin 10 µg/ml. The samples were separated on a Dionex Ultimate 3000 chromatography system equipped with a chromatographic column Acclaim C18 150×4.6, 5 μm. The mobile phase was a mixture of acetonitrile and water in a volume ratio of 90:10. Ivermectin and imidacloprid were detected at an absorption wavelength of 242 nm.
 Under the above-mentioned conditions, it was possible to completely separate imidacloprid and ivermectin (retention time of chromatographic peaks – 2.0 min and 10.0 min) and other components of the studied drug. At the same time, the suitability parameters of the chromatographic system did not exceed the limits specified in the recommendations of the USA Food and Drug Association. For the peaks of imidacloprid and ivermectin, the efficiency of the chromatographic system was 8000–10000 theoretical plates. The relative standard deviation (RSD) for the peak areas of the active substances was ± 0.31%, and the peak separation ratio (RS) of imidacloprid from ivermectin and other drug components was 35.9. The symmetry coefficient of the imidacloprid peak was 1.5, and that of ivermectin was 1.1. The calibration curves were linear in the recommended DFU 2.0 range (80–120% of the nominal concentration of the corresponding active substance). The coefficient of linearity (R2) for imidacloprid was 0.9991, and for ivermectin it was 0.9993.

Hereditary angioedema with normal C1-INH, developing recurrent acute abdomen after taking low-dose estrogen-progestin: A case report.

Hereditary angioedema is a rare genetic disease characterized by repeated episodes of temporary organ swelling. Three types of HAE are known, of which HAE with normal C1 inactivator is difficult to be diagnosed due to its lack of laboratory abnormalities. Here, we describe a case of HAE with normal C1 inactivator and recurrent acute abdomen following low-dose estrogen-progestin therapy. Notably, genetic analysis by Sanger sequencing led to the identification of a recurrent heterozygous missense mutation c.988A>G (p.K330E) in the plasminogen (PLG) gene of the patient. Prophylactic tranexamic acid and on-demand selective bradykinin B2 receptor blockers are used to treat her symptoms.

Role of tamsulosin a selective alpha 1a-1d blocker vs. combination therapy of tamsulosin and solifenacin M3 antagonist in DJ stent related morbidity: obstructive and irritative scores

Background: nowadays Urolithiasis is very common in general population. Due to the advent of newer lithotripsy techniques, endoscopic procedures are used in most of the cases and to promote stone clearance DJ stents are kept after all the endoscopic procedures. Methods: This is a prospective study conducted among indoor patients from January 2021 to May 2022 in 90 indoor patients of tertiary care hospital. Results: In our study go 90 patients, pre DJ insertion, IPSS score in group A was 10.22 and Group B was 9.64, Mean Quality of Life in Group A was 1.77 and group B was 1.37, Visual analogue scale in group A was 2.08 and group B was 2.24. On Day 1 Post DJ insertion, IPSS score in group A was 12.26 and Group B was 11.68, Mean Quality of Life in Group A was 1.44 and group B was 1.28, Visual analogue scale in group A was 2.55 and group B was 2.28. Conclusions: Use of combination of selective alpha 1a-1d blocker and M3 antagonist significantly reduces the DJ stent related morbidity after endoscopic treatment for urolithiasis.

Blockade of endothelin-1 receptor B regulates molecules of the major histocompatibility complex in sickle cell disease.

Major Histocompatibility Complex (MHC) molecules have been proposed to play a role in Sickle Cell Disease (SCD) pathophysiology. Endothelial cells express MHC molecules following exposure to cytokines. SCD is characterized, in part, by vascular endothelial cell activation, increased oxidative stress, sickle cell adhesion, and excess levels of endothelin-1 (ET-1) contributing to vaso-occlusive crises. ET-1 activates endothelial cells, induces oxidative stress and inflammation, and alters erythrocyte volume homeostasis. However, the role of ET-1 on MHC regulation in SCD is unclear. We first studied two sickle transgenic knockout mouse models of moderate to severe disease phenotype, βS-Antilles and Berkeley (BERK) mice. We observed significant increases in H2-Aa mRNA levels in spleens, lungs, and kidneys from transgenic sickle mice when compared to transgenic knockout mice expressing human hemoglobin A (HbA). Mice treated for 14 days with ET-1 receptor antagonists significantly reduced H2-Aa mRNA levels. We characterized the effect of ET-1 on MHC class II expression in the human endothelial cell line EA.hy926. We observed dose-dependent increases in the expression of MHC class II (HLA-DRA) and MHC transcription factor (CIITA) that were significantly blocked by treatment with BQ788, a selective blocker of ET-1 type B receptors. Chromatin immunoprecipitation studies in EA.hy926 cells showed that ET-1 increased Histone H3 acetylation of the HLA-DRA promoter, an event blocked by BQ788 treatment. These results implicate ET-1 as a novel regulator of MHC class II molecules and suggest that ET-1 receptor blockade represents a promising therapeutic approach to regulate both immune and vascular responses in SCD.

Design, synthesis, and mechanism of action of novel μ-conotoxin KIIIA analogues for inhibition of the voltage-gated sodium channel Nav1.7

μ-Conotoxin KIIIA, a selective blocker of sodium channels, has strong inhibitory activity against several Nav isoforms, including Nav1.7, and has potent analgesic effects, but it contains three pairs of disulfide bonds, making structural modification difficult and synthesis complex. To circumvent these difficulties, we designed and synthesized three KIIIA analogues with one disulfide bond deleted. The most active analogue, KIIIA-1, was further analyzed, and its binding pattern to hNav1.7 was determined by molecular dynamics simulations. Guided by the molecular dynamics computational model, we designed and tested 32 second-generation and 6 third-generation analogues of KIIIA-1 on hNav1.7 expressed in HEK293cells. Several analogues showed significantly improved inhibitory activity on hNav1.7, and the most potent peptide, 37, was approximately 4-fold more potent than the KIIIA Isomer I and 8-fold more potent than the wildtype (WT) KIIIA in inhibiting hNav1.7 current. Intraperitoneally injected 37 exhibited potent invivo analgesic activity in a formalin-induced inflammatory pain model, with activity reaching ∼350-fold of the positive control drug morphine. Overall, peptide 37 has a simplified disulfide-bond framework and exhibits potent invivo analgesic effects and has promising potential for development as a pain therapy in the future.

Reactive oxygen species produced by Zn2+ influx after exposure to AMPA, but not NMDA and their capturing effect on nigral dopaminergic protection

Glutamate excitotoxicity is involved in dopaminergic degeneration in the substantia nigra pars compacta (SNpc). Here we compared vulnerability to neurodegeneration after exposure to NMDA and AMPA. Apomorphine-induced movement disorder and dopaminergic degeneration in the SNpc, which are associated with Parkinson's syndrome, were induced after injection of AMPA into the SNpc of rats, but not after injection of NMDA. Co-injection of 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors rescued dopaminergic degeneration and increase in intracellular Zn2+ by AMPA. Furthermore, we tested the effect of capturing reactive oxygen species (ROS) produced by Zn2+ on neuroprotection in vivo. The levels of ROS, which were determined by HYDROP, a membrane-permeable H2O2 fluorescence probe and Aminophenyl Fluorescein (APF), a fluorescence probe for hydroxyl radical and peroxynitrite, were increased after injection of AMPA, but not after co-injection of CaEDTA, an extracellular Zn2+ chelator, suggesting that increase in Zn2+ influx by AMPA elevates the levels of intracellular ROS. AMPA-mediated dopaminergic degeneration was completely rescued by co-injection of either HYDROP or APF. The present study indicates that neurotoxic signaling of the influx of extracellular Zn2+ through Zn2+-permeable GluR2-lacking AMPA receptors is converted to ROS production and that capturing the ROS completely protects dopaminergic degeneration after exposure to AMPA, but not NMDA. It is likely that regulation of the conversion from Zn2+ influx into ROS production plays a key role to preventing Parkinson's syndrome.

Critical Pronociceptive Role of Family 2 Voltage-Gated Calcium Channels in a Novel Mouse Model of HIV-Associated Sensory Neuropathy.

Some people living with HIV present painful sensory neuropathy (HIV-SN) that is pharmacoresistant, sex-associated, and a major source of morbidity. Since the specific mechanisms underlying HIV-SN are not well understood, the aim of our study was to characterize a novel model of painful HIV-SN by combining the HIV-1 gp120 protein and the antiretroviral stavudine (d4T) in mice and to investigate the pronociceptive role of the family 2 voltage-gated calcium channel (VGCC) α1 subunit (Cav2.X channels) in such a model. HIV-SN was induced in male and female C57BL/6 mice by administration of gp120 and/or d4T and detected by a battery of behavior tests and by immunohistochemistry. The role of Cav2.X channels was assessed by the treatment with selective blockers and agonists as well as by mRNA detection. Repeated administration with gp120 and/or d4T produced long-lasting touch-evoked painful-like behaviors (starting at 6days, reaching a maximum on day 13, and lasting up to 28days after treatment started), with a greater intensity in female mice treated with the combination of gp120 + d4T. Moreover, gp120 + d4T treatment reduced the intraepidermal nerve fibers and well-being of female mice, without altering other behaviors. Mechanistically, gp120 + d4T treatment induced Cav2.1, 2.2, and 2.3 transcriptional increases in the dorsal root ganglion and the Cav2.X agonist-induced nociception. Accordingly, intrathecal selective Cav2.2 blockade presented longer and better efficacy in reversing the hyperalgesia induced by gp120 + d4T treatment compared with Cav2.1 or Cav2.3, but also presented the worst safety (inducing side effects at effective doses). We conclude that the family 2 calcium channels (Cav2.X) exert a critical pronociceptive role in a novel mouse model of HIV-SN.

Action potential metrics and automated data analysis pipeline for cardiotoxicity testing using optically mapped hiPSC-derived 3D cardiac microtissues.

Recent advances in human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues provide a unique opportunity for cardiotoxic assessment of pharmaceutical and environmental compounds. Here, we developed a series of automated data processing algorithms to assess changes in action potential (AP) properties for cardiotoxicity testing in 3D engineered cardiac microtissues generated from hiPSC-derived cardiomyocytes (hiPSC-CMs). Purified hiPSC-CMs were mixed with 5-25% human cardiac fibroblasts (hCFs) under scaffold-free conditions and allowed to self-assemble into 3D spherical microtissues in 35-microwell agarose gels. Optical mapping was performed to quantify electrophysiological changes. To increase throughput, AP traces from 4x4 cardiac microtissues were simultaneously acquired with a voltage sensitive dye and a CMOS camera. Individual microtissues showing APs were identified using automated thresholding after Fourier transforming traces. An asymmetric least squares method was used to correct non-uniform background and baseline drift, and the fluorescence was normalized (ΔF/F0). Bilateral filtering was applied to preserve the sharpness of the AP upstroke. AP shape changes under selective ion channel block were characterized using AP metrics including stimulation delay, rise time of AP upstroke, APD30, APD50, APD80, APDmxr (maximum rate change of repolarization), and AP triangulation (APDtri = APDmxr-APD50). We also characterized changes in AP metrics under various ion channel block conditions with multi-class logistic regression and feature extraction using principal component analysis of human AP computer simulations. Simulation results were validated experimentally with selective pharmacological ion channel blockers. In conclusion, this simple and robust automated data analysis pipeline for evaluating key AP metrics provides an excellent in vitro cardiotoxicity testing platform for a wide range of environmental and pharmaceutical compounds.

Pharmacological Profiling of KATP Channel Modulators: An Outlook for New Treatment Opportunities for Migraine.

Migraine is a highly disabling pain disorder with huge socioeconomic and personal costs. It is genetically heterogenous leading to variability in response to current treatments and frequent lack of response. Thus, new treatment strategies are needed. A combination of preclinical and clinical data indicate that ATP-sensitive potassium (KATP) channel inhibitors could be novel and highly effective drugs in the treatment of migraine. The subtype Kir6.1/SUR2B is of particular interest and inhibitors specific for this cranio-vascular KATP channel subtype may qualify as future migraine drugs. Historically, different technologies and methods have been undertaken to characterize KATP channel modulators and, therefore, a head-to-head comparison of potency and selectivity between the different KATP subtypes is difficult to assess. Here, we characterize available KATP channel activators and inhibitors in fluorescence-based thallium-flux assays using HEK293 cells stably expressing human Kir6.1/SUR2B, Kir6.2/SUR1, and Kir6.2/SUR2A KATP channels. Among the openers tested, levcromakalim, Y-26763, pinacidil, P-1075, ZM226600, ZD0947, and A-278637 showed preference for the KATP channel subtype Kir6.1/SUR2B, whereas BMS-191095, NN414, and VU0071306 demonstrated preferred activation of the Kir6.2/SUR1 subtype. In the group of KATP channel blockers, only Rosiglitazone and PNU-37783A showed selective inhibition of the Kir6.1/SUR2B subtype. PNU-37783A was stopped in clinical development and Rosiglitazone has a low potency for the vascular KATP channel subtype. Therefore, development of novel selective KATP channel blockers, having a benign side effect profile, are needed to clinically prove inhibition of Kir6.1/SUR2B as an effective migraine treatment.

Pericytes control vascular stability and auditory spiral ganglion neuron survival.

The inner ear has a rich population of pericytes, a multi-functional mural cell essential for sensory hair cell heath and normal hearing. However, the mechanics of how pericytes contribute to the homeostasis of the auditory vascular-neuronal complex in the spiral ganglion are not yet known. In this study, using an inducible and conditional pericyte depletion mouse (PDGFRB-CreERT2; ROSA26iDTR) model, we demonstrate, for the first time, that pericyte depletion causes loss of vascular volume and spiral ganglion neurons (SGNs) and adversely affects hearing sensitivity. Using an in vitro trans-well co-culture system, we show pericytes markedly promote neurite and vascular branch growth in neonatal SGN explants and adult SGNs. The pericyte-controlled neural growth is strongly mediated by pericyte-released exosomes containing vascular endothelial growth factor-A (VEGF-A). Treatment of neonatal SGN explants or adult SGNs with pericyte-derived exosomes significantly enhances angiogenesis, SGN survival, and neurite growth, all of which were inhibited by a selective blocker of VEGF receptor 2 (Flk1). Our study demonstrates that pericytes in the adult ear are critical for vascular stability and SGN health. Cross-talk between pericytes and SGNs via exosomes is essential for neuronal and vascular health and normal hearing.